Wet Adhesive Peptides

ABSTRACT

Peptides that form adhesive bonds, even in aqueous and/or saline environments, are disclosed. When aggregated, the peptides may be used in methods for producing hydrogels and/or adhesive materials. Synthetic peptide analogs are provided that are designed based on protein sequences found in barnacle adhesive, and may optionally be augmented with chemistry from other organisms that secrete proteins that adhere to substrates. The peptides may be used, for example, in biomedical and aqueous applications. Methods of using the aggregated peptides as adhesives are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.17/163,733 filed on Feb. 1, 2021 which itself is a is a division of U.S.patent application Ser. No. 16/182,425 filed on Nov. 6, 2018 (now U.S.Pat. No. 10,927,148), which in turn claims priority to U.S. ProvisionalPatent Application No. 62/582,022 filed on Nov. 6, 2017, the entirety ofeach of which is incorporated herein by reference.

SEQUENCE LISTING

This application contains a Sequence Listing, which has been submittedelectronically in ASCII format and is hereby incorporated by referencein its entirety.

FIELD OF THE DISCLOSURE

The invention relates to peptides that form adhesive bonds, even inaqueous and/or saline environments. When aggregated, the peptides may beused in methods for producing hydrogels and/or adhesive materials.Synthetic peptide analogs are provided that are designed based onprotein sequences found in barnacle adhesive, and may optionally beaugmented with chemistry from other organisms that secrete proteins thatadhere to substrates. The peptides may be used, for example, inbiomedical and aqueous applications. Methods of using the aggregatedpeptides as adhesives are also provided.

BACKGROUND OF THE INVENTION

Smart materials, wherein chemistry and material properties respond toenvironmental cues, are beneficial in applications where materials mustbe formed upon delivery, e.g., at a bonding surface or a wound site. Incontrast to covalently bound materials such as cyanoacrylates, thesematerials can reversibly undergo phase transitions at ambienttemperatures in response to external chemical or mechanical cues.

Fibrous biomaterials such as biological amyloids are of particularinterest due to their high degree of order and unified molecularstructure. They have become a desirable class of biomaterials whereproperties of a simple primary sequence can scale across molecular,nano-, meso-, and macroscopic distances. Knowles, et al. recentlydemonstrated these materials can be processed to form ordered bulk filmswith moduli of 5-7 GPa, rivaling films made from rigid carbon nanotubes,and consistent with the modulus of a single amyloid nanofiber (Knowles,T. P. J., et al., J. Nature Nanotechnology 2011, 6, 469).

X-ray diffraction and molecular modeling indicate that most naturalamyloid materials are composed of crystalline β-sheets. The pattern ofamino acids in these structures allows the placement of chemical specieswith high spatial precision solely through a primary letter sequence.For example, peptides with an alternating leucine (L) sequence motifwill form micron-long nanofibers in which X side chains are displayedwhile leucine groups are buried (Rufo, C. M., et al., Nat Chem 2014, 6,303). Thus, properties at the macroscopic length-scale are determined bya distinct sequence-structure relationship, forming tightly folded betasheet structures that both maintain a strong network of fibers andpromote display of functional amino acid side chain chemistries. Thoughmuch work has been done to understand the hierarchy of physicalproperties in templated and self-assembled amyloid materials, impartingchemistry and multiple functions to these materials presents anunexplored frontier.

Despite intense efforts to develop temperature, pH and ionic strengthtriggers in protein and peptide materials, copolymerization of two ormore distinct components as a smart material trigger has received littleattention. For example, peptides designed with hairpin turns fold into aprecursor structure that is favorable for fiber assembly when specificside-chains are deprotonated under basic conditions (Schneider, J. P.,et al., J Am Chem Soc 2002, 124, 15030; Yucel, T., et al.,Macromolecules 2008, 41, 5763). Other synthetic proteins are based onelastins (Brennan, M. J., et al., Biomaterials 2017, 124, 116; McMillan,R. A., et al., Macromolecules 2000, 33, 4809; Huang, L., et al.,Macromolecules 2000, 33, 2989), which are triggered by exploiting aglass transition temperature dependent on the arrangement of thesequence.

Current synthetic materials rely on temperature, pH and ionic strengthto form bulk gels, while many natural systems achieve transitionswithout external cues purely by recognition between proteins (So, C. R.,et al., Sci Rep 2016, 6, 36219; Barnhart, M. M., et al., Annu RevMicrobiol 2006, 60, 131; Chapman, M. R., et al., Science 2002, 295, 851;Hammer, N. D., et al., P Natl Acad Sci USA 2007, 104, 12494; Cheung, P.J., et al., Mar Biol 1977, 43, 157; Kamino, K., Biofouling 2013, 29,735). Engineered systems are largely homopolymers, formed from a singlemonomer, limiting the additional functionality that could be broughtabout by the co-polymerization of two or more components. Thesesequences also involve more than 50% of the chain chemistry in theassembly and formation of fibers, providing little room forincorporation of other functionalities.

In contrast to marine organisms that use glues to fabricate protectiveshelters (e.g., sand-castle worm tubes, case-maker fly larva retreats,and amphipod tubes) or tie themselves to rocks, (e.g., mussel byssusthreads) adult barnacles produce their adhesive interface in asequential process hidden under their base as a part of their normalgrowth cycle. The recent finding that barnacle adhesive isnanostructured and held together as an amyloid-like material furtherdistinguishes it from archetypal marine adhesives processed into solidfoams or spun threads. The permanent adhesive produced by adultbarnacles is held together by tightly folded proteins that formamyloid-like materials unique among marine foulants. The adhesive ispolymerized from protein subunits that form a micron-thick layer ofordered nanofibers and function as a permanent wet adhesive.

Barnacles use hydrogen bonds to tightly fold their adhesive proteins anddisplay side chains to achieve enhanced mechanical strength, proteinbundling, and co-localized reactive chemistries. The polymerization ofbarnacle glue occurs through these molecular interactions, and theresulting adhesive is a meshwork of nanoscale fibers. Proteomic surveyssuggests that barnacle glue functions by displaying adhesive chemistriesthrough small and flexible side-chains, folded in a manner similar toadhesive silks used by spiders and insects (So, C. R., et al., Sci Rep2016, 6, 36219). These fibers are complex co-polymers, formed from thespecific interactions between more than 20 different protein components.Their well-defined, modular, nature permits barnacle adhesive to servemany purposes: adhesion, durability, bacterial resistance, and evenpotent enzymatic activity (So, C. R., et al., ACS Appl Mater Interfaces2017, 9, 11493). Moreover, the absence of cellular processes at theattachment surface suggests that delivery, assembly, and displayedchemistry of adhesive nanomaterials are governed by the physicalproperties of patterned non-charged amino acids.

Hydrogels and adhesives have been developed based on various peptides.For example, U.S. Patent Appl. Publ. No. 2017/0015885 by Liu et al.(“Liu”) is directed to protein-based adhesives. An elastin-likepolypeptide is provided.

U.S. Pat. No. 7,884,185 to Schneider et al. (“Schneider”) is directed tohydrogels and methods of making and using such hydrogels. Schneiderprovides hydrogels that may be formed by the self-assembly of peptidesin solution. Such self-assembly may be brought about by a change in oneor more characteristics of the solution. Characteristics of the solutionthat may be changed include pH, ionic strength, temperature, andconcentration of one or more specific ions. The hydrogels described bySchneider may be disassembled by changing one or more characteristic ofthe hydrogel.

U.S. Pat. No. 9,228,009 to Hartgerink et al. (“Hartgerink”) is directedto collagen, and more particularly compositions and methods related tocollagen-mimetic peptides. Hartgerink provides a collagen-mimeticpeptide and peptide systems.

U.S. Pat. No. 9,493,513 to Mehmet et al. (“Mehmet”) is directed topolypeptides that bind to inorganic solid surfaces, structurescomprising such polypeptides, and methods of making such structures.

However, there remains a need in the art for peptide-derived polymersand hydrogels, particularly those that may be used as adhesives inaqueous environments.

SUMMARY OF THE INVENTION

The invention described herein, including the various aspects and/orembodiments thereof, meets the unmet needs of the art, as well asothers, by providing peptides that form adhesive bonds, even in aqueousand/or saline environments. When aggregated, the peptides may be used inmethods for producing hydrogels and/or adhesive materials. Syntheticpeptide analogs are provided that are designed based on proteinsequences found in barnacle adhesive, and may optionally be augmentedwith chemistry from other organisms that secrete proteins that adhere tosubstrates. The peptides may be used, for example, in biomedical andaqueous applications. Methods of using the aggregated peptides asadhesives are also provided.

In one aspect of the invention, a peptide is provided that has an aminoacid sequence of any one of SEQ ID Nos:1-8.

In another aspect of the invention, a peptide is provided that has anamino acid sequence including one of the conserved sequence patterns ofSEQ ID Nos:11-20.

Peptides can be further tailored to include chemistries that enhanceadhesive properties, through addition of amino acids or modification ofexisting amino acids.

According to a further aspect of the invention, a method for forming awet adhesive includes providing a first surface; covering at least aportion of the first surface with a first peptide; and introducing asecond peptide in contact with the first peptide. Upon contact of thefirst peptide with the second peptide, the first peptide and secondpeptide self-assemble into a solid adhesive material.

Other features and advantages of the present invention will becomeapparent to those skilled in the art upon examination of the followingor upon learning by practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a plot of charged residues per sequence for a multiplesequence alignment of five barnacle cement proteins, which show 10instances of a 19 kD motif repeated along full length protein sequences.

FIGS. 2A-2E show sequence-dependent polymerization of dormant and activeBCPs. FIG. 2A shows a glue shaving from Amphibalanus amphitrite exposedto ThT (10 mM) for <15 min and imaged via a fluorescence microscope,with an inset untreated glue shaving. FIG. 2B shows that ThT increasesfluorescence intensity upon binding to BCP2C fibrils present insolution. FIG. 2C shows that synthetic barnacle cement peptides havevaried activity over a 300 hour period. FIG. 2D shows that activeBCP1C/2/2C in Tris-EDTA and ASW undergo a growth phase, reaching asteady state ThT fluorescence. FIG. 2E shows the estimated aggregationonset (Lag time (Tin_(g))).

FIG. 3 shows the nano and microstructure of formed BCP fibrils from ThTscreening. The top image corresponds to the full-length protein MRCP19forming short fibers that clump together. The image on the left showsthe point mutated mutBCP1 (G19C) peptide demonstrating classicalnucleation, lag phase and growth, also forming short fibrils thataggregate, adjacent to (right) BCP1C, containing an additionalneighboring charged domain to result in fibers that are 5+ microns inlength. The next image to the right shows BCP2, with only a simpledomain, forming a similar fiber structure as shown for MRCP19, whereshort assemblies clump to form larger particles. The image furthestright shows BCP2C fibrils, including an additional neighboring patternedcharge domain that forms 5+ micron long fibrils with a discrete meshedarchitecture.

FIGS. 4A and 4B show amide regions from transmission FTIR of dried BCPmaterials on CaF₂ that displayed activity by ThT. Both spectra show aprominent Amide I mode at ca. 1625 cm⁻¹ indicative of an amyloid-likematerial, similar to the parent barnacle glue. FIG. 4A shows, from topto bottom, BCP2 with only a simple domain maintains similar modes butdoes not display the prominent feature at 1698 cm⁻¹. BCP2C spectrumshowing three additional modes at 1525 cm⁻¹ (Amide II), 1661 cm⁻¹ and1698 cm⁻¹ (anti-parallel turn). mutBCP1 amide region similar to BCP2with a main peak at 1626 cm⁻¹ and a shoulder at 1656 cm⁻¹ with no modesnear 1700 cm⁻¹. Bottom, BCP1C spectrum showing similar modes as theBCP2C spectrum including the prominent shoulder at 1698 cm⁻¹. FIG. 4Bshows peak deconvolution of amide modes from FIG. 4A, showing aprominent peak at 1698 cm⁻¹, using five element Lorentzian fit forBCP2/2C/1C and three element fitting for mutBCP1.

FIGS. 5A-5E show that the patterned charge domain in BCP2C confersmolecular recognition by templating dormant BCPs to form fibrillarstructures. Cross-seeding assay histograms are presented of preformedactive BCP fibrils incubated with free BCPs in both Tris-EDTA (FIG. 5A)and ASW (FIG. 5B) are presented. Dormant BCP activation is defined as aThT fluorescence≥100 a.u. The cross-seeding assay shows BCP2C to havethe ability to activate most dormant peptides, which BCP2 and BCP1C donot showing the recognition of seeds is sequence specific. Seeds alonehave a low intrinsic fluorescence indicated by *. Dormant peptideactivation predominantly occurs with BCP2C exogenous seeds.Characteristic amyloid seeds of Aβ42 fibrils show no activation of BCPs.FIGS. 5C-5E show representative ThT fluorescence aggregation curves ofseed activation or acceleration. Fit of sigmoidal least squaresregression trace (solid black line) shown with data points. Values oflag time are obtained from this fit. FIG. 5C shows that dormant BCP3C isactivated in the presence of preformed BCP2C seed fibrils in Tris-EDTAfrom no measured onset to 17 h. FIG. 5D shows the preformed BCP2Cradically accelerated BCP2 aggregation onset from 30 h to <2 h. FIG. 5Eshows that dormant BCP1 is activated in the presence of preformed BCP1Cseed fibrils in ASW from no onset to 30 h.

FIGS. 6A-6C show that randomization of BCP2C modifies peptide activityand aggregation by disruption of distinctive hydrophobic core sequence.FIG. 6A is a schematic representation of BCP2C that demonstrates thesimple and charged domains, as well as an identified hydrophobic core inboth the sequence and hydropathy. FIG. 6B shows the amino acid sequenceof BCP2C randomized (ranBCP2C) with corresponding hydropathy. In bothFIG. 6A and FIG. 6B, a 5-window moving average of hydropathy using theKyte and Doolittle scale is overlaid as a line with a scale of −3.5 to1.5, where hydrophilic residues have a hydropathy index below zero andhydrophobic resides above zero. The grand average of hydropathy (GRAVY)value of both BCP2C and ranBCP2C was −0.7378. FIG. 6C shows thatranBCP2C inhibits self-assembly (no activity) and polymerization after300 hours.

FIGS. 7A-7C show the incorporation of BCP peptides in newly depositedbarnacle cement. FIG. 7A shows re-settled barnacles grown onnitrocellulose membranes (NC) for 72 hours. FIG. 7B shows a Western blotanalysis of barnacles peeled from NC, where the left panel represents amembrane incubated with HRP conjugated anti-rabbit antibodies. Thecenter panel and right hand side panels are independent re-settledbarnacles blotted against α-CP43 and α-CP19, respectively. FIG. 7C showsNC-bound barnacle cement incubated with (from left to right) FITClabeled BCP4 (positive response), TRITC labeled BCP2C (positiveresponse) or TRITC labeled poly-lysine peptide (negative response).

FIG. 8 shows the mechanism of dormant peptide activation throughmolecular recognition. Two classical sigmoidal curves are involved inthe recognition process, indicating that the full physical aggregationand growth mechanism occurs first from the self-assembling peptide(I-III) and secondly from the dormant peptide recognizing the nascentstructured active peptide template (IV-VI). Inset fluorescencemicroscopy of TRITC labelled BCP2C seeds exposed to FITC labelled BCP4free peptide show coincident red and green fluorescence, indicative of alayer-by-layer growth of BCP4 onto pre-existing BCP2C seeds.

FIG. 9 shows raw and normalized absorbance data from FTIR as a ratio of1625 cm⁻¹ to 1698 cm⁻¹.

FIG. 10 Seed assay performed using mutBCP1 and Aβ42 seeds against BCPs3/4/3C/4C showing little cross-seeding activity.

FIG. 11 is a Kyte and Doolittle hydropathy plot of BCP1C showinghydrophobic stretch across residues 7-11.

DETAILED DESCRIPTION OF EMBODIMENTS OF THE INVENTION

The invention described herein, including the various aspects and/orembodiments thereof, meets the unmet needs of the art, as well asothers, by providing peptides that form adhesive bonds, even in aqueousand/or saline environments. When aggregated, the peptides may be used inmethods for producing hydrogels and/or adhesive materials, includingmaterials that exhibit adhesive properties underwater. Synthetic peptideanalogs are provided that are designed based on protein sequences foundin barnacle adhesive, and may optionally be augmented with chemistryfrom other organisms that secrete proteins that adhere to substrates.The peptides may be used, for example, in biomedical and aqueousapplications. Methods of using the aggregated peptides as adhesives arealso provided.

In the context of the present invention, barnacles include organisms ofthe order Sessilia, preferably barnacles of family Balanidae(particularly barnacles of the genera Balanus, Fistulobalanus, andAmphibalanus), and family Archaeobalanidae (particularly barnacles ofthe genus Semibalanus). The peptides of the invention may be derivedfrom the cement of Amphibalanus amphitrite, a species of acorn barnacle.These sequences exhibit a unique dual nature that is both an orderedsilk-like structure and a chemical adhesive, as non-charged segmentsdemonstrate homology (e-value of ca. 10-30) with adhesive silk motifs.Well-defined sequence domains, as observed in barnacles, provide thepresent invention with the ability to exert independent control overstructural and adhesive properties. In some aspects of the invention,this can be achieved by utilizing a sequence of approximately 35-50residues, preferably approximately 35-40 residues.

A family of barnacle proteins rich in distinct stretches of alternatingcharged and non-charged linear sequence has been identified inaccordance with the invention. These stretches are referred to as“charged” domains, where not more than 50% of the sequence is comprisedof charged amino acids, and not more than 50% of the sequence iscomprised of non-charged amino acids. Recombinant protein and peptidesequences are designed using the patterned domains identified in thenatural adhesive that contain non-charged and charged sequence[Gly/Ala/Thr/Ser/Val-X], where X corresponds to charged amino acids.These sequences are referred to as barnacle cement derived peptides(BCPs). Charged amino acids relevant to the invention are Arginine (R),Lysine (K), Aspartic acid (D), Glutamic acid (E), Histidine (H).Non-charged amino acids relevant to the invention are Serine (S),Threonine (T), Glycine (G), Alanine (A), Leucine (L), Isoleucine (I),Valine (V). Charged domains alternate with stretches of non-chargedamino acids, referred to as “simple” domains. Simple domains arecomprised of mostly non-charged amino acids as defined above, with notmore than 20% charged amino acid content. The role of simple and chargeddomains in forming fibrillar materials has also been identified. Thesesequences exert specific control over timing, structure and morphologyof fibril formation.

While most BCPs remain dormant, a core segment demonstrates rapidpolymerization as well as an ability to template other peptides, even ifthey have no intrinsic propensity for self-assembly. Without wishing tobe bound by theory, it is believed that the patterned charge domainsassemble dormant peptides through a unique anti-parallel beta sheetstructure. While charged domains favor an anti-parallel structure, BCPswithout charged domains switch fibril assembly to favor simpler parallelbeta sheet aggregates. In addition to activation, charged domains addpersistence length to resulting nanofibers, mimicking fibrils observedin the natural adhesive, while segments without such domains only formshort, branched aggregates. The specific activation of dormant peptidesthrough recognition of structured amyloid-like templates is similar tomechanisms of biological recognition between proteins, and permitscontrol over physical mechanisms including adhesive delivery,activation, and curing.

Peptides.

The invention provides peptide sequences that can be polymerized to formhydrogels and adhesives. The sequences of the invention contain a highlyconserved pattern alternating between 20-25 peptide-long, “simple”domains having a greater proportion (80-100%) of non-charged amino acids(i.e., Gly, Ser, Thr, Ala, Val, Ile residues), and 10-25 peptide-long,“charged” domains having a greater proportion (40-60%) of charged (i.e.,Arg/Lys/Asp/Glu/His residues) intermixed with not more than 50%non-charged amino acids.

The sequences of the invention form distinct patterns that result instructural fibers. Five core proteins identified in the cement ofbarnacles (Accession ID numbers AKZ20819.1, AQA26371.1, AQA26372.1,AQA26373.1, and AQA26376.1) each comprise from 9 to 25 of these shortalternating simple and charged domains, underscoring their role as amajor design element for marine adhesion. The peptides of the inventionmay therefore be provided as short sequences including a simple domainand a charged domain. These simple and charged domains may be repeatedto form longer synthetic peptides incorporating from 6-100 domains,preferably from 8-60 domains, more preferably from 10-30 domains. Thesame simple and charged domains may be repeated as a pattern throughoutthe synthetic peptides, or several different simple and charged domainsof the invention may be used to form the synthetic peptides. The simpleand charged domains may be provided without any intervening amino acidsbetween each domain, or intervals of several amino acids may separatethe domains.

These simple domain and charged domain sequence patterns are conservedamong Amphibalanus amphitrite barnacle cement proteins. As shown in FIG.1 , charged and simple domains were identified based on a multiplesequence alignment of barnacle cement proteins from A. amphitrite,specifically, by alignment of the proteins corresponding to Accession IDnumbers AKZ20819.1, AQA26371.1, AQA26372.1, AQA26373.1, and AQA26376.1.A 19 kD domain was identified in Accession ID No. AKZ20819.1 thatspanned amino acids 33-199, which was found to correspond to similardomains in Accession ID numbers AQA26371.1 (a first domain wasidentified that spanned amino acids 31-204, and a second domain wasidentified that spanned amino acids 205-368); AQA26372.1 (a first domainwas identified that spanned amino acids 55-220, and a second domain wasidentified that spanned amino acids 228-393), AQA26373.1 (a first domainwas identified that spanned amino acids 40-214, and a second domain wasidentified that spanned amino acids 254-442), and AQA26376.1 (threedomains were identified: a first domain that spanned amino acids 21-182,a second domain that spanned amino acids 195-360, and a third domainthat spanned amino acids 385-518). The charged residues are plotted inthe domain map shown in FIG. 1 .

These identified sequence patterns were used to design a set of peptidesthat contain conserved, non-charged (termed, “simple”) domains alongwith their neighboring patterned, charged (termed, “charged”) domains.These synthetic mimics are referred to as barnacle cement peptides(BCPs).

The simple domains include, but are not limited to:

BCP1 (SEQ ID NO: 1) QTGYTRGGAAVSSTGATQGAGS BCP2 (SEQ ID NO: 2)AVGNSGVSGSGVSIGDSGFRQKTQT BCP3 (SEQ ID NO: 3) TGTQGKGITSGEAVANQKAGAEGGBCP4 (SEQ ID NO: 4) GTSSSGHKASSSGPGRFITSNThe simple domains further include peptides having 90% or greateridentity to these sequences, preferably 95% or greater identity to thesesequences, more preferably 98% or greater identity to these sequences.

The combined simple and charged domains include, but are not limited to:

BCP1C (SEQ ID NO: 5) QTGYTRGGAAVSSTGATQGAGS LDLAIDGPGGFKARSK BCP2C(SEQ ID NO: 6) AVGNSGVSGSGVSIGDSGFRQKTQT NSEAGSKGTKRA BCP3C(SEQ ID NO: 7) TGTQGKGITSGEAVANQKAGAEGG AQRVEAVKYVESDGKNLYKVEKVD BCP4C(SEQ ID NO: 8) GTSSSGHKASSSGPGRFITSN EVGTEIKLTTPELDThe combined simple and charged domains further include peptides having90% or greater identity to these sequences, preferably 95% or greateridentity to these sequences, more preferably 98% or greater identity tothese sequences.

In addition to these synthetic peptides, a mutant form of BCP1 and arandomized form of BCP2C were also generated in order to compare theirproperties with those of the sequences set forth above. These peptidesinclude:

mutBCP1 (SEQ ID NO: 9) QTGYTRGGAAVSSTGATQ(G→C)AGS ranBCP2C(SEQ ID NO: 10) GKRSGQDGTTGSGNVSETSSSFVKGKAAVGRGQINSA

Also within the scope of the invention are sequences that incorporateconserved patterns along the length of the 19 kD homologous sequence.These peptide patterns include:

(SEQ ID NO: 11) [QxGxTGGAxVSxxGxTQGxGS]_(simple) +[patterned charged domain]_(charged) (SEQ ID NO: 12)[AVGNSGVSGxGxSxGxGxFxQ]_(simple) + [patterned charged domain]_(charged)(SEQ ID NO: 13) [VTxTxGxGxTxGxAxQKAGANGG]_(simple) +[patterned charged domain]_(charged) (SEQ ID NO: 14)[AxSSSGHxASSxGxGxFxVxNxxxTExK]_(simple) +[patterned charged domain]_(charged) (SEQ ID NO: 15)[TxTxGxGxTGxAxxxQKAGANGG]_(simple) +[patterned charged domain]_(charged) (SEQ ID NO: 16)[xTSSSGHxASSxGxGxFxVxN]_(simple) + [patterned charged domain]_(charged)

In some aspects of the invention, the conserved peptide patterns may befurther characterized as follows:

(SEQ ID NO: 17) [QxGxTxGGAxVSxxGxTQGxGS]_(simple) +[xDxxxDGGGGDKxRxK]_(charged) (SEQ ID NO: 18)[AVGNSGVSGNGxSxGxGxFxQ]_(simple) + [xxExxxKxxKRx]_(charged)(SEQ ID NO: 19) [VxTYTxGxGxTxGxAxxxQKAGANGG]_(simple) +[xxRxExxKxxExDxKxxxKxEKxD]_(charged)  (SEQ ID NO: 20)[AxSSSGHxASSxGxGxFxVxNxxxTExK]_(simple) + [ExxxExKxxxxExD]_(charged)

In each of these conserved peptide patterns, the [x] denotes apermissive site that is not conserved. In some aspects of the invention,each instance of “x” independently corresponds to an amino acid selectedfrom the group consisting of Gly, Ser, Thr, Ala, Val, Ile, Lys, Arg,Glu, Asp, Asn, Gln residues. Within a given peptide, each x may be thesame or different. Preferably, x is an amino acid selected from thegroup consisting of Gly, Ser, Thr, Ala, Val, Ile.

For example, the patterned charged domain used in the peptide patternfor BCP1C may encompass the following peptides, in which [x] correspondsto Ala, Ser, Gly, or Thr:

(SEQ ID NO: 21) QAGATAGGAAVSAAGATQGAGS ADAAADGGGGDKARAK (SEQ ID NO: 22)QSGSTSGGASVSSSGSTQGSGS SDSSSDGGGGDKSRSK (SEQ ID NO: 23)QGGGTGGGAGVSGGGGTQGGGSGDGGGDGGGGDKGRGK (SEQ ID NO: 24)QTGTTTGGATVSTTGTTQGTGS TDTTTDGGGGDKTRTKHowever, the invention is not limited to these particular peptides, andencompasses peptides in which [x] corresponds to a residue selected fromGly, Ser, Thr, Ala, Val, Ile, Lys, Arg, Glu, Asp, Asn, or Gln, whereeach instance of [x] may be the same or different.

For example, the patterned charged domain used in the peptide patternfor BCP2C may encompass the following peptides, in which [x] correspondsto Ala, Ser, Gly, or Thr:

(SEQ ID NO: 25) AVGNSGVSGAGASAGAGAFAQ AAEAAAKAAKRA (SEQ ID NO: 26)AVGNSGVSGSGSSSGSGSFSQ SSESSSKSSKRS (SEQ ID NO: 27)AVGNSGVSGGGGSGGGGGFGQGGEGGGKGGKRG (SEQ ID NO: 28)AVGNSGVSGTGTSTGTGTFTQ TTETTTKTTKRTHowever, the invention is not limited to these particular peptides, andencompasses peptides in which [x] corresponds to a residue selected fromGly, Ser, Thr, Ala, Val, Ile, Lys, Arg, Glu, Asp, Asn, or Gln, whereeach instance of [x] may be the same or different.

For example, the patterned charged domain used in the peptide patternfor BCP3C may encompass the following peptides, in which [x] correspondsto Ala, Ser, Gly, or Thr:

(SEQ ID NO: 29) VATATAGAGATAGAAAAAQKAGANGG AARAEAAKAAEADAKAAAKAEKAD(SEQ ID NO: 30) VSTSTSGSGSTSGSASSSQKAGANGG SSRSESSKSSESDSKSSSKSEKSD(SEQ ID NO: 31) VGTGTGGGGGTGGGAGGGQKAGANGG GGRGEGGKGGEGDGKGGGKGEKGD(SEQ ID NO: 32) VTTTTTGTGTTTGTATTTQKAGANGG TTRTETTKTTETDTKTTTKTEKTDHowever, the invention is not limited to these particular peptides, andencompasses peptides in which [x] corresponds to a residue selected fromGly, Ser, Thr, Ala, Val, Ile, Lys, Arg, Glu, Asp, Asn, or Gln, whereeach instance of [x] may be the same or different.

For example, the patterned charged domain used in the peptide patternfor BCP4C may encompass the following peptides, in which [x] correspondsto Ala, Ser, Gly, or Thr:

(SEQ ID NO: 33) AASSSGHAASSAGAGAFAVANAAA TEAK EAAAEAKAAAAEAD(SEQ ID NO: 34) ASSSSGHSASSSGSGSFSVSNSSS TESK ESSSESKSSSSESD(SEQ ID NO: 35) AGSSSGHGASSGGGGGFGVGNGGG TEGK EGGGEGKGGGGEGD(SEQ ID NO: 36) ATSSSGHTASSTGTGTFTVTNTTT TETK ETTTETKTTTTETDHowever, the invention is not limited to these particular peptides, andencompasses peptides in which [x] corresponds to a residue selected fromGly, Ser, Thr, Ala, Val, Ile, Lys, Arg, Glu, Asp, Asn, or Gln, whereeach instance of [x] may be the same or different.

The peptides of the invention may exhibit varying levels of fibrilformation, and different sets of peptides may be created to tailor theextent of fibril formation and the rate of fibril formation. Preferably,one set of peptides is limited to the simple domains (i.e., BCP1, BCP2,BCP3, and BCP4) while the other set of peptides includes both the simpledomain and an additional charged domain incorporating G-x, S-x, and/orV-x sequence patterns (where x is as defined above, and designatescharged residues). Examples of charged domains include those found inthe patterned domains incorporated into the peptides BCP1C, BCP2C,BCP3C, and BCP4C.

The peptides of the invention may be provided individually, or ascombinations of two or more of the peptides of the invention. Thepeptides may be provided together or separated into sets of peptides.The first peptide or first set of peptides may be dormant (i.e., theymay not self-assemble into fibrils), and may only assemble into fibrilson contact with a second peptide or second set of peptides, where thesecond peptide or second set of peptides templates the first peptide orfirst set of peptides to cause fibril formation. The first and secondpeptides may self-assemble into fibrils that result in adhesion whenapplied to surfaces. In some aspects of the invention, the first set ofpeptides includes peptides having an amino acid sequence as set forth inany one of SEQ ID Nos:1-8 and 11-20. In further aspects of theinvention, the second set of peptides is selected from peptides havingan amino acid sequence based on those set forth for BCP2 and BCP2C (SEQID Nos:2, 6, 12, and 18).

The peptides of the invention may further be chemically altered byintroducing new amino acids to the sequence (e.g., at the N and/or Cterminal, or between one or more repetitions of the simple and chargeddomains of the invention) or modifying existing amino acids to augmentadhesive properties underwater. This can be done by replacing, forexample, non-charged sequence positions with tyrosine residues andperforming oxidation of the side chain to dihydroxyphenylalanine (DOPA).DOPA has been shown to displace water and form bidentate interactionswith inorganic surfaces. Alternatively, serines in the existing sequencecan be modified by chemical or biochemical means to display phosphategroups, which mediate wet adhesion through divalent ions and ionicinteractions with surfaces. Serines can also be modified with long orshort chain polysaccharides, which promote adhesive interactions withinorganic surfaces. These modifications all enhance chemical adhesion ofthe formed material in underwater environments.

The peptides may be screened using high throughput tools for amyloidcharacterization including fluorometric Thioflavin T (ThT) experiments,fluorescence microscopy, atomic force microscopy (AFM), and FourierTransform Infrared Spectroscopy (FTIR) of formed materials. (See, e.g.,FIGS. 2A-2E and 3A-3C.) Certain simple peptide motifs aggregate and formfibrils, while the addition of one or more neighboring charged patternstemplate microns-long fibril materials of higher order. The addition ofcharged domains can template other sequences with no ability to formfibrils on their own, rendering them able to form fibrils as a result.This templating capability of certain peptides of the invention providesa strategy for timed polymerization in the delivery and curing of anadhesive in an aqueous environment.

The peptides according to the invention may be chemically-produced usingknown methods of peptide synthesis, for example by solid phasesynthesis. The peptides may also be produced using recombinant methodsthat rely upon introducing genes encoding the peptides of interest intoa host organism that is suitable for production of the peptides, and thehost organism transcribing and translating the genes. The genes may beintroduced into a host organism via a vector, in particular anexpression vector. The functional unit including a gene, anoperably-linked promoter, and optionally other genetic elements arereferred to as an “expression cassette.”

Nucleotides that encode the peptides of the invention may be formedusing methods which are generally known in the art, such as chemicalsynthesis or the polymerase chain reaction (PCR), in conjunction withstandard molecular biology and/or protein synthesis techniques. Forexample, it is possible for those skilled in the art, on the basis ofknown DNA and/or amino acid sequences, to produce the correspondingnucleic acids and even complete genes.

The invention further provides prokaryotic or eukaryotic cells that havebeen transformed with nucleic acids that encode for peptides accordingto the invention.

Hydrogels and Adhesive Materials.

Adhesive materials in accordance with the invention preferably exhibitproperties that include: (1) they exist as a liquid prior to bonding,and solidify at the site to be adhesively bonded; (2) the formedmaterial spans two surfaces (i.e., a first surface and a second surface,which may have the same or different compositions); (3) the formedmaterial provides enhanced cohesive bond strength by way of networkedcovalent or non-covalent polymer chemistries; and (4) the formedmaterial displays chemistry capable of displacing water and formingchemical interactions with surfaces in aqueous environments. Preferredmaterial systems of the invention exhibit all four of these propertiessimultaneously. Synthetic analogs to natural barnacle glue sequences canbe tailored to display enhanced or shielded underwater adhesiveinteractions with surfaces. These non-natural peptides, used in whollysynthetic environments, are demonstrated to fulfill all criteria as anunderwater adhesive material system as embodied in this invention.

The peptides of the invention may be used to produce hydrogels that canbe delivered as a liquid, and subsequently polymerized by recognition ofBCP2/BCP2C and related peptides to form an adhesive gel. These peptidesrepresent a new class of peptide hydrogel materials, wherecopolymerization occurs specifically between designed sequences to formheterogeneous smart gel materials.

The invention provides hydrogels that comprise networked barnacle-likeprotein fibers held together by organized hydrogen bonds exhibit highrigidity at low weight percentages, yet retain the ability to revertback to liquid form under mechanical shear. The hydrogels have theability to be externally triggered into gelation, back to liquid form,and reassembled into a gel network. The ability for the inventiveadhesive materials to be delivered to the bonding site as a liquid, andtriggered to polymerize into a functional gel is based on non-covalentinteractions and recognition between unique protein sequences.

Specific amino acid sequences found in barnacle cement proteins controlmaterial polymerization and curing underwater through specificintermolecular interactions and aggregation timescales, providing ameans to localize adhesive formation. The short synthetic peptides ofthe invention mimic segments of smart adhesive gels from barnacles, andalso demonstrate control over polymerization, where a unique mastersequence (i.e., BCP2/BCP2C and related peptides) induces fiber formationand gelation in dormant sequences. Lock-and-key recognition by syntheticbioinspired peptides is therefore a viable strategy for the delivery andformation of smart gel materials with adhesive properties. The controlover polymerization and delivery of adhesive gels underwater usingbarnacle cement-mimicking peptides is beneficial for biomedical andmarine adhesives, as well as applications that requireenvironmentally-responsive hydrogel adhesives.

Formation of smart gels by sequence recognition and co-polymerization ofdesigned peptide sequences into bulk barnacle cement-derived peptidematerials allows the incorporation of multiple functions, includingunderwater adhesion, into a deliverable system. Incorporation ofmultiple components into a co-polymer via sequence recognition alsoallows control of component order found along composite fibers. Thisdiscovery sets barnacle-derived peptides apart from existing designedpeptide-based materials. Imparting multiple chemical and biologicalfunctions to materials is currently a multistep process, requiringspecific surface and protein linker chemistries as well as reactionsthat work only in dry environments. Marine organisms such as barnacles,on the other hand, produce a chemically-, biologically-, andmechanically-robust attachment to nearly any surface by depositing justone single layer of proteinaceous material. These robust materials arestructured as a meshwork of ordered nanoscale fibers, formed inseawater, and survive years of harsh marine exposure. The syntheticpeptides of the invention also achieve these benefits.

Adhesive compositions in accordance with the invention may be formed byincorporating the inventive peptides into a vehicle, such as an aqueousvehicle, along with one or more buffers, crowding agents such aspolysaccharides (Ficoll®, GE Healthcare, Chicago, Ill.) and polyethyleneglycol (PEG), glycerol, sea water or artificial sea water (InstantOcean®, Instant Ocean Spectrum Brands, Blacksburg, Va.), preservatives,or other additives as known to those skilled in the art.

Adhesive compositions in accordance with the invention may also beformed by incorporating the inventive peptides into a water immisciblevehicle, which may include one or more nonpolar solvents (including, butnot limited to, dimethyl sulfoxide, dimethyl formamide, anddichloromethane), lipids, and oils, and optionally including additionalcrowding agents such as polysaccharides (Ficoll®) and polyethyleneglycol (PEG), preservatives, or other additives as known to thoseskilled in the art.

The adhesives of the invention may be provided as a single solution, oras two separate solutions to be combined when the adhesive bond isformed. When provided as a single component, a shear force may beapplied to the peptides of the invention in order to deliver thepeptides to a surface being bonded. Once the shear force is removed andthe peptides are in place on the surface, the peptides self-assembleinto fibrils. When provided as a two-component system, a first peptideis applied to a surface being bonded, where the first peptide is notcapable of self-assembly into fibrils. The application of the secondpeptide causes the first peptide to be templated, and results inself-assembly of the mixture of first and second peptides into fibrils.

Conventional adhesives may be replaced by the inventive peptides inorder to provide adhesives capable of being used in environments thatare moist or aqueous, including plant and animal tissues, freshwaterstructures and vehicles, and saltwater/marine structures and vehicles.In addition to being specifically adapted for use in aqueousenvironments, they are also suitable for use in applications whereconventional adhesives are used. In additional aspects of the invention,the peptide-based adhesives of the invention may be used in conjunctionwith conventional adhesives.

Assays.

The invention also provides assays, which may be used by those skilledin the art to determine if a particular peptide is likely to havecementitious properties, or be useful as a hydrogel and/or adhesive. Toestablish experimental conditions that form fibers from designed mimics,assays are provided that are sensitive to ordered molecular structures.The assays have the ability to monitor growth kinetics for up to 96simultaneous reactions spanning 48 hours, while maintaining a constantvolume in the hundred-microliter range.

This assay technique may be used to monitor solution state kineticsexhibited by distinct solution phases (monomeric, oligomeric,protofibril and mature fiber) of well-studied amyloid proteins duringfiber formation, as well as altered modes exhibited by the peptides ofthe invention (FIG. 4A-4B). This assay enables a rapid characterizationof positive and negative responding candidates, which informsexperimental design criteria such as pH, ionic strength, and peptideconcentration that can overcome energy barriers for fiber nucleation. Asan example, exposure of positively responding mimics to a chargedmineral surface and subsequent imaging by atomic force microscopy (AFM)(FIG. 5A-5E) confirms an assembled glue-like fiber structure, andhighlights their ability to form dense mat-like ultrastructures similarto bulk barnacle glue at the liquid-solid interface.

Assays of the invention include antibodies capable of binding to thepeptide sequences of the invention. The antibodies preferably areprovided along with one or more markers capable of binding to theantibody-peptide complex, and providing an indication that such bindinghas occurred. The indicator may be a visual indicator, a chemicalindicator, a biological indicator, a radioactive indicator, or any otherform of indicator known in the art.

The assays may be carried out using any suitable substrate. Onepresently-preferred substrate is a 96-well plate, but those skilled inthe art will appreciate that the assays of the invention may be used inconjunction with other assay techniques and apparatus.

Methods.

The bioinspired nanofibers of the invention, which resemble thestructure of amyloid materials, can bring multiple functions to asurface with a single deposition step. The invention establishes newmethods of functionalizing surfaces, particularly wet surfaces orsurfaces in an aqueous environment, where specific sequences polymerizeto form gels and coatings that simultaneously add adhesive, mechanical,and advanced chemical or biological functionalities to diverse materialsurfaces.

The sequences and bioinspired nanofibers of the invention may be usedfor applications that include, but are not limited to, aqueousapplications, undersea applications, and biomedical applications. Newadhesives suitable for use in these and other aqueous environments areneeded.

The peptides of the invention may be used in methods for forming anadhesive bond. One or more first peptides are applied to at least aportion of a first surface to be adhesively bonded to a second surface.The adhesive bond is then formed by placing a second peptide in contactwith the first peptide. The second peptide may be placed in contact withthe first peptide by any suitable technique. In some instances, this mayoccur by applying it to at least a portion of the second surface, andthen contacting the first peptides on the first surface with the secondpeptides on the second surface. When the first peptide and the secondpeptide come into contact, the first and second peptides self-assembleinto fibrils that result in adhesion between the first and secondsurfaces. In some aspects of the invention, the first peptide isselected from peptides having an amino acid sequence based on those setforth in any one of SEQ ID Nos:1-8 and 11-20. In further aspects of theinvention, the second peptide is selected from peptides having an aminoacid sequence based on those set forth in BCP2 and BCP2C (SEQ ID Nos:2,6, 12, and 18).

Depending on the particular surfaces to be adhesively joined, the firstsurface may be provided in an aqueous environment, although theinvention is not limited to the use of the adhesives in aqueousenvironments. When the first surface is in an aqueous environment, theaqueous environment may be a body of water, such as an ocean, sea, lake,or river, and the first surface may be a component of an underwaterstructure, such as a dock or offshore rig, or seagoing vessel, such as aship or submarine.

The first surface may be also be a tissue of a plant or animal. Plantand animal tissues may be adhesively joined in order to repair wounds.In accordance with methods for adhesively joining tissue, the firstpeptides may be applied to a first tissue surface, and then joined withsecond peptides applied to a second tissue surface. When the peptidesare brought together, the second peptides cause the formation of fibrilsthat result in adhesion between the first and second tissue surfaces.

In situ polymerization is also used to form injectable gels for tissuerepair or as tissue adhesives and sutures in wet wound environments.Since BCP-based materials are held together by non-covalent bonds,hydrogels can be delivered as shear-thinned liquids that thenre-assemble into gels at their injection site. This opens alternativetechnology avenues in developing injectable biomedical adhesivematerials.

The peptides of the invention may be altered chemically by introducingnew amino acids to the sequence or modifying existing amino acids toaugment adhesive properties underwater. This can be done by replacing,for example, non-charged sequence positions with tyrosine residues andperforming oxidation of the side chain to dihydroxyphenylalanine (DOPA).DOPA has been shown to displace water and form bidentate interactionswith inorganic surfaces. Alternatively, serines in the existing sequencecan be modified by chemical or biochemical means to display phosphategroups that mediate wet adhesion through divalent ions and ionicinteractions with surfaces. Serines can also be modified with long orshort chain polysaccharides, which promote adhesive interactions withinorganic surfaces. These modifications all enhance chemical adhesion ofthe formed material in underwater and/or saline environments.

EXAMPLES

The invention will now be particularly described by way of example.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the invention. Thefollowing descriptions of specific embodiments of the present inventionare presented for purposes of illustration and description. They are notintended to be exhaustive of or to limit the invention to the preciseforms disclosed. Many modifications and variations are possible in viewof the above teachings. The embodiments are shown and described in orderto best explain the principles of the invention and its practicalapplications, to thereby enable others skilled in the art to bestutilize the invention and various embodiments with various modificationsas are suited to the particular use contemplated.

Example 1. Peptide Synthesis

Designer peptides were prepared on an automated solid-phase peptidesynthesizer employing standard stepwise Fmoc protection and deprotectionchemical procedures. Synthesis was carried out on a preloadedFmoc-Val(tBu)-Wang low-loading support resin using HBTU activationchemistry, while 20% piperidine in DMF was employed to afford the Fmocdeprotection, monitored by UV absorbance at 301 nm. Peptides werecleaved off the support and side chain deprotected by stirring theresin-bound peptide in a cocktail containing 90:5:3:2TFA/thioanisole/EDT/anisole under an N₂ atmosphere for ˜6 h. Peptideswere precipitated in cold ether and collected on vacuum filtrationmembrane, and lyophilized overnight. Purification of products wascarried out by reverse-phase HPLC, where the most prominent peak wasanalyzed by MALDI-MS. Peptides and proteins were used as freeze-driedproduct and self-assembled at 1 mM concentrations at pH 8 in phosphatebuffer to produce fibers.

Full length MRCP19 from Megabalanus rosa (acorn barnacle) was cloned andexpressed recombinantly by GenScript USA.

Example 2. Peptide Preparation

Lyophilized, synthetic barnacle derived peptides were prepared accordingto published protocols of β-amyloid. In short, the peptides were treatedwith 200 μL hexafluoroisopropanol (HFIP) and sonicated (10 min) todissolve preexisting or seed aggregates within the lyophilized stock.HFIP was evaporated off using a speedvac (Labconco), resulting in clearpeptide films. These peptide films were dissolved in varying amounts ofdimethyl sulfoxide (DMSO) to make a 10 mM stock solution of each BCP andsonicated (10 min). Stock solutions were stocked at −80° C. to preventaggregation. To achieve the desired concentration, the stock solutionswere dissolved directly into 50 mM Tris-HCl, 1 mM EDTA (Tris-EDTA)buffer, or sterilized Artificial Sea Water (ASW) with a salinity of 32ppm. From the 10 mM stock solution of BCP1C/2/2C, 200 μM aggregated orseed solutions of these active (fibril forming) BCP peptides wereprepared in their corresponding buffer (Tris or ASW) at 37° C. withorbital shaking for 48-96 hours, over 7-10 days.

Prepared fibrillar seeds were purified and concentrated in ˜200 μLdesired buffer for use in seeding assays. BCP2 and 2C formed largeaggregates which were pelleted using a MiniSpin Plus (14.1 rcf, 30-60min) (Eppendorf), decanted, and reconstituted in Tris-EDTA, ASW, ordeionized water accordingly to 20 μM. For BCP2C, the presence ofdiscrete fibrils inhibited pelleting and seeds were concentrated andused directly from stock concentrations. In addition, β-amyloid 1-42(A1342) (Genscript USA, Inc., Piscataway, N.J.) 10 mM stocks in DMSOwere prepared using the same amyloid protocol above and seed solutionswere formed over 300 hours and concentrated in Tris EDTA and ASW.Concentrated seed BCPs and seed A1342 were sonicated for 10-15 minimmediately before use and ThT fluorescence of the seeds was checkedprior to use in seeding assays. BCP2 underwent additional sonication(30% amplitude pulse for 5 min) with a sonic horn (Qsonica LLC, Newtown,Conn.) to ensure complete homogenization of BCP2 seeds. BCP1C/2/2C andmutBCP1 were used in seeding assays to activate non-fibril formingpeptides in aggregation assays described below. A1342 seeds were used tosee if non-active BCPs had specificity to amyloid-forming materials.

Example 3. Characterization of Peptide Polymerization

A stock solution of 1 mM ThT was prepared and a working solution of 100μM ThT was used in each assay. Two different ThT aggregation assays wereperformed: 1) Free peptide BCP and 2) Seeding from either BCP1C/2/2C orA1342 with free peptide BCP. In the free peptide BCP assay, each BCP(BCP1-4, BCP1C-4C, mutBCP1, and ranBCP2C) were prepared individually andThT fluorescence measured at various concentrations (200 μM, 100 μM, and50 μM) of each synthetic peptide. In addition to free peptide BCP, ThTassays were also performed with full length mrcp19 (1×) synthesizedrecombinantly (Genscript USA, Inc., Piscataway, N.J.). In the seeded BCPassay, concentrated ˜20 μM seed solutions of active (fibril forming)BCP1C/2/2C and A1342 were placed with dormant (non-fibril forming) BCP1/3/3C/4/4C at 200 μM. Both ThT assays were incubated with 100 μM ThT at32° C. in Grenier black bottom 96-microwell plates (Sigma-Aldrich) thatwere sealed to prevent evaporation. ThT fluorescence was measured every15 min for up to 300 h using a Synergy H1 Hybrid Multi-mode reader(BioTek, Winooski, Vt.) with excitation and emission filters set at 440and 480 nm, respectively with a low gain of 50, linear shaking, and topread. All ThT fluorescence assays were performed in duplicate (or more)on each plate and maximum fluorescence or normalized fluorescence wasplotted in arbitrary units (a.u.). ThT assay results were verified amongthree or more independently performed experiments on additional 96 wellplates. All kinetic data from ThT aggregation curves (free peptide BCPand seeded BCP assays) at each respective concentration were fit with anon-linear (sigmoidal) least squares regression to obtain estimatedaggregation onset values/lag time (T_(lag)).²⁴

${F(t)} = \frac{F_{0} + A}{\left( {1 + e^{- {k({t - t_{\frac{1}{2}}})}}} \right)}$

Fitted parameters of the least squares regressions are k (elongationrate constant), A (amplitude), F₀ (baseline), and T_(1/2) (time at halfcompletion of aggregation). As defined by Hellstrand, et al. T_(lag) wascalculated from the fitted parameters as:

$T_{Lag} = {T_{1/2} - \frac{2}{k}}$

Once modelled, the maximum fluorescence of the least squares fit foreach sample was obtained. These maximum fluorescence measurements of twoor more individual samples under identical experimental conditions (i.e.same concentration, seed preparation, temperature) were averaged andreported with standard deviation.

Example 4. Nanostructure Characterization by Atomic Force Microscopy

For ex situ AFM imaging, 20 μL samples from identified 96 microwells ofinterest were spotted onto freshly cleaved muscovite mica and placed ina hood to allow for evaporation of buffer to dryness (12-24 h). Each drysample was washed with aliquots of deionized water and dried under agentle stream of nitrogen. A Digital Instruments (Santa Barbara, Calif.)Dimension 3100 scanning probe microscope equipped with high frequencyNanoSensors PPP-NCHR (NanoandMore USA, Lady's Island, S.C.) probes witha 42 N/m spring constant was used to image peptide nanostructures. Allimaging was carried out under tapping mode, with 512×512 dataacquisitions at a scan speed of 0.8 Hz at room temperature underacoustic isolation. Supplier-provided software (Nanoscope, V7.3, Veeco)was utilized for extracting quantitative data such as surface crosssections from AFM images. After images were collected, average featurewidths were quantified by measuring the peak-to-peak distances over thespan of multiple fibers.

Example 5. Observation of Fiber Formation

To establish experimental conditions that form fibers from designedmimics, a fluorometric assay was developed that is sensitive to orderedmolecular structures, and has the ability to monitor growth kinetics forup to 96 simultaneous reactions spanning 48 hours while maintaining aconstant volume in the hundred microliter range. This technique is usedto monitor solution state kinetics exhibited by distinct solution phases(monomeric, oligomeric, protofibril, and mature fiber) of well-studiedamyloid proteins during fiber formation, as well as altered modesexhibited by the designed peptide mimics. This assay enables a rapidcharacterization of positive and negative responding candidates, whichinforms experimental design criteria such as pH, ionic strength, andpeptide concentration that can overcome energy barriers for fibernucleation. As an example, exposure of positively responding mimics to acharged mineral surface and subsequent imaging by atomic forcemicroscopy (AFM) (FIGS. 3A-3C) confirms an assembled glue-like fiberstructure, and highlights their ability to form dense mat-likeultrastructures similar to bulk barnacle glue at the liquid-solidinterface.

Of the surveyed sequences, only BCP2C is observed to polymerize and formnanofibers with the length and morphology of native glue fibers (compareFIG. 3C with FIGS. 3A and 3B). BCP1 and BCP2 also respond to ThT (FIGS.3B and 3C), forming fibrils that display a short and bundled morphology.BCP1, BCP3, BCP3C, BCP4, and BCP4C display no activity and are referredto as dormant (FIG. 2C). BCP2C therefore is a likely candidate as a coresequence in forming native barnacle glue. To prove that this sequence iscentral in forming fibers, purified BCP2C fibers were exposed to BCPsthat display dormant activity. In the presence of BCP2C seeds, shown inFIG. 10 , higher ThT activity as well as more rapid polymerization ofdormant BCPs over exposure to seeds prepared from BCP1 and BCP2 wereobserved. Therefore, the BCP2C sequence is found uniquely capable ofactivating other peptides to induce polymerization via molecularrecognition.

Example 6. Barnacle Husbandry

Amphibalanus amphitrite barnacles were settled as cyprids onsilicone-coated glass panels and reared at the Duke University MarineLaboratory (Beaufort, N.C.) as described previously (So, C. R. et al.,“Oxidase Activity of the Barnacle Adhesive Interface InvolvesPeroxide-Dependent Catechol Oxidase and Lysyl Oxidase Enzymes,” ACS ApplMater Interfaces 9(13):11493-11505 (2017)). Panels of adult barnaclesgrown to 2-3 mm in diameter were shipped to the Naval ResearchLaboratory (Washington, D.C.), where they were maintained in anincubator operating at 23° C. on a 12 h day/night cycle in 32 ppt ASW(Instant Ocean, Blacksburg, Va.). The barnacles were fed Artemia spp.nauplii (Brine Shrimp Direct, Ogden, Utah) three times a week, and theASW was changed once a week during which excess algal growth wasremoved. Barnacles used for experiments were gently dislodged from thesilicone-coated panels, rinsed with distilled water, and placed onalternative substrates for the experiments.

Example 7. Immunoblotting of Barnacle Cement

Adult barnacles were placed onto a nitrocellulose membrane (0.45 μm),fed and housed in ASW at room temperature for 72 hour settlement toallow for resettlement via cement deposition (FIG. 7A). After 72 hours,barnacles were gently peeled off (n=3) and the resultant membraneunderwent Western immunoblotting. The membrane was blocked in 5% non-fatmilk dissolved in 1×PBS-T, pH 7.4, (10 mM phosphate buffered saline,0.05% Tween 20) for 1 hour, washed three times with PBS-T and probedwith 1:1,000 dilution of anti-CP43 or anti-CP19 rabbit (FIG. 7B). Thecorresponding anti-rabbit HRP conjugated secondary antibody was used at1:10,000 dilution (FIG. 7A). Membranes were developed using the WesternDura chemiluminescence kit (Pierce) and image acquisition via Gel DocXR+ Gel Documentation System (Bio-Rad, Hercules, Calif.). Antibodieswere generated by GenScript USA.

Example 8. Activity of Labeled BCP Towards Natural Glue Secretions

Adult barnacles were settled onto nitrocellulose membrane (0.45 pm)similar to immunoblotting experiments above. After 3 days, barnacleswere peeled off (n=6) and the resultant membrane was blocked in 5%non-fat milk dissolved in 1×PBS-T, pH 7.4. Blocked membranes were washedthree times in PBS-T and incubated overnight in 10 μM solutions offluorescently labelled free BCPs, dormantBCP4-fluorescein-5-isothiocyanate (FITC) (max excitation 490 nm, maxemission 525 nm) and active BCP2C-tetramethylrhodamine isothiocyanate(TRITC) (max excitation 557 nm, max emission 576 nm) (FIG. 7C). Inaddition, preformed BCP2C-TRITC seeds (100 μM) were formed at 37° C.with orbital shaking over 7-10 days. As negative controls, a solution ofa TRITC-labeled peptide (GGGRDGGG) was incubated with membrane depositedbarnacle glue (FIG. 7C). Each incubation was repeated with multipleanimals (n=3). Fluorescence microscopy images were collected on a NikonA1R+ laser scanning confocal microscope to detect free and seeded BCPsover areas with transferred adhesive.

Example 9. Unique Core Sequences are Responsible for Polymerization ofMaterials

To verify that amyloid-like barnacle adhesive responds to Thioflavin T(ThT), exposed untreated glue shavings (a.k.a., gummy glue) were takendirectly from the barnacle Amphibalanus amphitrite to ThT. ThT is acationic benzothiazole dye that probes for beta sheet rich structuresresponsible for holding amyloid fibrils together. Shown in FIG. 2A,natural glue shavings demonstrate enhanced ThT fluorescence responseafter a 15-30 minute incubation at room temperature, verifying that thenatural adhesive responds to dyes sensitive to classical amyloids inagreement with previous studies highlighting the amyloid-like structureusing FTIR and CD.

The formation of amyloids is commonly classified as anucleation-dependent polymerization reaction with a characteristicinitial lag phase, followed by an elongation/growth phase during whichmost fibrils form, and ending in a plateau of no further amyloid fibrilincrease. The physical states of aggregation can be classified based ona classical sigmoidal curve produced as ThT-responsive materials formover time in solution, including stages of fibril growth that spansmonomers, oligomers, protofibrils, and mature fibrils. To determinephysical properties of representative patterned BCPs, all eight peptideswere surveyed by exposing them to ThT solution over the course of up to300 hours. Of the BCPs, three distinct peptide sequences (BCP1C/2/2C)demonstrate the characteristic sigmoidal curvature associated withfibril formation (FIG. 2D) and a positive ThT response (maximumfluorescence) (FIG. 2C) in both simple buffer and seawater conditions.In contrast, BCPs 1/3/3C/4/4C displayed no activity or fibril formationover 300 h, and are referred to as dormant BCP sequences (FIG. 2C). Inboth Tris-EDTA and artificial seawater (ASW), BCP2 shows the highestactivity, followed by BCP2C and BCP1C which both contain chargeddomains. All three active peptides responded and underwent fibrilformation in an artificial sea water environment with similar onsettimes as in Tris-EDTA, largely unaffected by free ions present in ASW.The lag time (T_(lag)) is the time at which aggregation begins, definedas an x-intercept extrapolated from the rising linear portion of thesigmoid. Seen in FIG. 2E, T_(lag) values were obtained using a sigmoidalmodel fitted by least squares regression of each experimental curve. ThTfluorescence curves (FIG. 2D) and estimated aggregation onset times(FIG. 2E) reveal that BCP2/2C exhibit faster aggregation onset timeswith T_(lag) values within ca. 12 h in Tris-EDTA and ca. 50 h in ASW. Incontrast to BCP1, BCP1C is considerably slower with a delayedaggregation time of 100+ hours. In Tris-EDTA, BCP2C noticeably had theshortest aggregation time with the lowest spread of T_(lag) (2-16 h,median 6 h), followed by BCP2 (4-53 h, median 26 h), while BCP1Cexhibited the longest aggregation time (64-208 h, median 120 h). BCP2C/2revealed a large T_(lag) spread ranging from 2-102 h (median 22 h) and9-73 h (median 30 h) respectively. BCP1C remained the slowest with anonset of 101-180 h (median 169 h). The aggregation onset of fibrillarformations follows the trend BCP2C<BCP2<BCP1C for both Tris-EDTA and ASWwith BCP1C being significantly delayed.

Example 10. Charged Patterns Confer Folded, Microns-Long FibrilMorphology to Simple Sequences

To understand the effect of charged domains on the morphology andmolecular structure of peptide fibrils, ThT responsive materials werecharacterized by AFM and FTIR (FIGS. 3 and 4A-4B, respectively). Clearmorphological and structural differences were observed between simplepeptides alone (BCP1/2) and peptides that also contained a variablecharged sequence (BCP1C/2C). This is highlighted in FIG. 3 , wheresimple peptides assemble into short branched structures spanning 1-2microns, similar to the full length protein, while their chargedcounterparts assemble into features that span 5+ microns. The differencebetween BCP2 and BCP2C is especially stark, where the charged sequenceconfers a matted discrete structure while the simple peptide aloneclumps as aggregates from shorter subunit fibrils. Whole length 19 kDprotein from Megabalanus rosa (MRCP19), containing all homologousdomains, forms very short fibers that do not exceed 1 micron which isconsistent with other observations. Since BCP1 showed no ability toassemble on its own, a single gly→cys mutation (G19C, mutBCP1) wasintroduced to promote intermolecular interactions through disulfidebonds. Indeed, like the other active peptides, a single mutation inmutBCP1 induced classical aggregation, lag and fiber growth phases whenthe solution was monitored using ThT. Fibers formed by mutBCP1 aresimilar to those of another simple peptide, BCP2, in that fibers remainin the 1-2 micron range, yet are 5+ microns in the presence of theneighboring charged domain as seen in BCP1C and 2C.

Example 11. Transmission Infrared Spectroscopy of Peptide NanofibersDried on CaF2 Reveal Clear Distinctions in the Secondary Structure ofSequences that Contain Patterned Charge Domains

Shown in FIGS. 4A-4B, all fibril materials formed by peptides absorbstrongly in the Amide I region, with the largest peak centered at ca.1623 cm⁻¹ typical of tightly folded beta-strand secondary structures.Two other prominent features range from 1655-1660 cm⁻¹ for all peptidefibrils as well as a prominent shoulder at 1698 cm⁻¹ for BCP1C/2C. Thebroad Amide I feature at ca. 1660 cm⁻¹ is also present in amyloid beta.However, the unique mode at 1698 cm⁻¹ observed in conjunction with thenarrow mode observed at ca. 1625 cm⁻¹ indicates that peptides BCP1C andBCP2C form beta strands oriented in an anti-parallel fashion. Incontrast, fibrils made from peptides without charged domains (mutBCP1and BCP2) do not display the 1698 cm⁻¹ mode, consistent with a parallelbeta sheet structure. Quantification of the maximum peak absorbance at1698 cm⁻¹ as a fraction of the peak at ca. 1625 cm⁻¹ highlights largedifferences in the two vibrational modes between charged and non-chargedpeptides. Peptides with charged domains BCP2C and BCP1C have fractionsin the 0.9-1.0 range (when normalized to 1), while the absence of thefeature at 1698 cm⁻¹ causes BCP2 and mutBCP1 to exhibit low fractionsbetween 0.15 and 0.3 (FIG. 9 ). These data suggest that patterned chargedomains confer an anti-parallel structure to formed fibrils.

Example 12. Patterned Charge Domains Activate Dormant Barnacle CementPeptides Through Molecular Recognition

Since charged and non-charged domains appear in a sequential order alongthe natural protein sequences (FIG. 1 ), the ability of organized seedstructures to serve as templates in activating peptides from downstreamregions was tested. To this end, the ability of BCP fibrils (BCP1C/2/2C)to polymerize dormant BCPs (BCP1/3/3C/4/4C) or accelerate kinetics offibril formation was probed using a cross-seeding ThT reaction in bothTris-EDTA (FIG. 5A) and ASW (FIG. 5B). For this, BCP seeds were formedover 7-10 days in Tris-EDTA, purified and incubated with free peptidesolutions. Fibrillar BCP seeds alone have a low inherent ThTfluorescence over 300 h. Activation of dormant peptides was measured andcounted only if the fluorescence exceeded the minimum response of activeBCPs, i.e., ≥100 a.u. Interestingly, while BCP2 and mutBCP1 form fibrilsas free peptides, they lack an ability to stimulate elongation andactivation of dormant BCPs (FIG. 10 ). Seeds from peptides withoutcharged domains have little activity, while seeds containing chargeddomains initiate the polymerization of most dormant peptides in bothTris-EDTA and ASW. The charged domain present in both BCP1C and BCP2C,as well as their unique anti-parallel secondary structure, adds acomponent of recognition for dormant peptide sequences not observed inmutBCP1 or BCP2. This robust recognition establishes BCP2C as the mostself-active, as well as activity inducing, sequence of the eightpeptides studied. This is exemplified in FIG. 5C, where dormant peptideBCP3C is placed in a cross-seeding assay with preformed fibrils ofBCP2C. While no measured onset over 300 h is observed (FIGS. 2C and 2D)for dormant BCP3C alone, the peptide polymerizes in the presence ofBCP2C seeds in under 17 h. Furthermore, seeding by BCP2C not onlyactivates most dormant peptides, but also accelerates the formation ofamyloid fibrils in active peptides such as BCP2. The lag phase forcross-seeded reactions of active free BCP2 was <2 h in the presence ofBCP2C seeds, compared to 25-30 h for unseeded reactions (FIG. 5D).

Interestingly, BCP1C is observed to only activate peptides BCP1 and 4 inASW (FIG. 5B). Similar to BCP2C, BCP1C contains both a simple andcharged domain and is shown to template polymerization of its simpledomain (BCP1) counterpart. While BCP1 alone has no measured onset over300 h, it readily polymerizes at 30 h in the presence of BCP1C seeds(FIGS. 2C and 2D). Thus, peptides displaying a patterned charge domainare uniquely capable of inducing downstream dormant peptides to formamyloid fibers, possibly due to the anti-parallel configuration offibers. BCPs were then exposed to a well-known amyloid seed organized ina parallel beta sheet structure, human beta-amyloid 1-42 (AB42), todetermine whether this response is specific to barnacle adhesives. Inthese assays, no ThT response (FIGS. 5A and 5B) was observed with AB42fibrils, showing that polymerization is specific to barnacle gluematerials and the unique secondary structures formed by alternatingcharged/non-charged sequences.

Example 13. Randomization of BCP2C Sequence Eliminates Propensity forPolymerization and Recognition

To test the importance of the linear sequence in both the polymerizationand activation of downstream sequences, the BCP2C sequence wasrandomized, and ThT activity and aggregation activity was monitored(FIGS. 6A and 6B). Upon randomization, the N-terminal hydrophobic coresequence and corresponding amphiphilic nature were disrupted, shiftingthe C-terminus of the peptide to become more hydrophobic (FIG. 6B).Interestingly, ranBCP2C showed no ThT activity over 300 h (FIG. 6C),showing that it could no longer polymerize into fibrillar structureslike that of its predecessor BCP2C, and that a sequence withoutamphiphilic property or an alternating charge pattern can no longerundergo recognition and assembly.

Example 14. BCPs Bind to Naturally Secreted Barnacle Adhesive

The ability for short BCP sequences to recognize naturally secretedadhesive from A. Amphitrite acorn barnacles was tested. Antibodiesgenerated against adhesive proteins as well as two fluorescently labeledBCPs were used as probes against nitrocellulose membranes wherebarnacles have been resettled for three days. The presence of AA19 andAA43, two proteins in the adhesive abundant in non-charged amino acids,were first probed to verify that they had been transferred to themembrane surface. FIG. 7 shows a strong response to the primaryantibodies, with little or no background response compared to thesecondary antibody alone. Negative controls with luminol, a peroxidaseactivated chemiluminescent substrate, showed little cross reactivitywith endogenous peroxidases known to exist in the barnacle adhesive.Both antibodies reacted strongly to regions where the organism resided,where proteins are seen distributed either localized to the barnacleperiphery where new growth occurs or filled into the center of thesettled region. A second set of membranes were probed with BCP2C andBCP4 peptides labelled with TRITC and FITC, respectively. A negativecontrol peptide containing a GGGKDGGG sequence sensitive to oxidases wasused, which showed no activity towards the glue region. Both BCP4 aswell as BCP2C displayed strong fluorescence when exposed to thetransferred glue region, demonstrating that there is an element ofrecognition for BCPs by the natural glue materials.

DISCUSSION

The unique primary sequence of BCP2C enables it to serve as a coreaggregation domain, form extended fibril structures similar to barnacleglue, and activate most other dormant peptide sequences in bothTris-EDTA and ASW. Since fibril formation is largely unaffected bysolution ionic strength, hydropathy trends in the BCP2C sequence wereexamined (FIG. 6A). Much like in amyloid beta and designed beta sheetstructures, hydrophobic sequences may play a central role in folding,activation, and self-assembly of BCP2C fibrils. The 37 residue sequenceof BCP2C contains both a simple, non-charged domain (BCP2) where threealiphatic residues V7, V12, and 114 form an 11-residue hydrophobicsegment (average hydropathy >3) that abruptly transitions to a chargedand hydrophilic C-terminal region. Aggregative hydrophobic domains suchas those found in BCP2 may also be ‘gated’ by alternating chargedresidues, which can explain the lack of polymerization in BCP1 and longdelayed onset of nanofiber formation in BCP1C. BCP1 contains only onecentral aliphatic valine, while BCP1C contains two leucines and anisoleucine alternating with two aspartic acids in a 5-residue stretch(FIG. 11 ). These three aliphatic residues may be prevented frominteracting due to negative charge repulsion from neighboring asparticacid side chains, adding substantial time to achieve necessaryaggregation.

These examples demonstrate that BCPs derived from patterned barnaclecement proteins undergo a process of induced fit occurring throughreversible interactions, principally structured hydrogen bonding, toform complex nanomaterials as they first exist as free peptides and onlybecome active in material formation when induced by a uniqueanti-parallel folded template. Two classical sigmoidal curves wereobserved during the activation process: one for the active sequence anda subsequent curve for the nucleation and growth of dormant peptides.The observation of a second complete curve indicates that dormantpeptides undergo the full physical process of fiber assembly in thepresence of structured templates. This mechanism is outlined in FIG. 8 ,where the activation of a dormant peptide follows a typicalprotein-protein recognition scheme: I) Free peptides exist in solutiondisplaying little structure, II) peptides coalesce and assemble intonucleates, III) nucleates condense into structured beta-sheet fibrils,IV) structured peptides from III are now recognized by free dormantpeptides, V) dormant peptides undergo nucleation and typical sigmoidalgrowth from existing seeds, and finally VI) nucleates collapse intobeta-sheet structures to form multicomponent fibrils. As protein foldingbegins with nearest neighbor interactions, two pathways would lead toBCP2C existing in an active folded structure: first, BCP2 beinghydrophobic would induce the neighboring patterned charge domain to foldinto an anti-parallel configuration within the same protein. Thisstarting structure then templates the folding of subsequent sequencesinto a fiber comprised of 19-like proteins. Secondly, since peptides areobserved to complement with the natural adhesive, homologous BCP2domains from two proteins could condense through hydrophobicinteractions to allow neighboring charged domains in BCP2C to fold andinitiate the activation process of downstream domains in the sameprotein or among multiple proteins.

Although BCPs are abundant in charged residues, a consistent finding isthat polymerization occurs similarly in both artificial seawater andsimple buffers or DI H₂O, solutions of diverse ionic strength. Thisimplies that charged residues play a minor role in forming fibers,emphasizing a mechanism where fibrils are formed through flexiblenon-charged side-chains and hydrogen bonds, similar to other fibrousbiomaterials. Assembly and formation of underwater materials throughsimple hydrophobic aggregation would allow the organism to operate inseawater and form materials in the presence of metal ions. Opposite tohighly aggregative domains, the ability for certain domains tocompletely resist polymerization indicates that there also existso-called “gatekeeping” domains which act as a delay for proteins toform materials. Although BCP2C and BCP2 polymerize rapidly, certaindomains took over 100 hours, or 3-4 days, to become active even in thepresence of a BCP2C template. This indicates that even in partiallyfolded and activated states, there exist segments which delay the curingprocess of glue components on the timescale of real barnacle molting andgrowth processes.

Fibrillar amyloid structures used by barnacles are unique among marinefoulants, yet they bear similar alternating sequences to specializedprotein fibrils used by arthropods and crustaceans. These includenanofibrous adhesive protein produced by distantly related spiders,pyriform spidroin. These materials resemble barnacle glue, as they existas an embedded nanofibrous meshwork that envelops a central dragline.Pyriform spidroin proteins do not exhibit the conventional subrepeatmotifs found in spider fibroin, rather they display regions ofalternating polar and nonpolar amino acids, similar to the barnacleadhesive. Two examples include the primary repeated region as well asthe more variable region from the primary pyriform spidroin proteinPySpl. The non-charged residues in the latter motif are polymorphic,similar to the observed pattern in barnacle adhesive proteins. Further,S-X and G-X motifs in silk fibroin proteins directly relate to theability to produce turns in shaping the threads produced by silk worms.

The alternating sequences present in barnacle adhesive induce foldingwhen in tandem with conserved segments that exist in at least 10locations across multiple proteins. Spiders, moths and silk-producingcrustaceans are closer relatives to barnacles than the well-studiedadhesives of bivalves such as mussels and tube worms, which bear nosignificant chemical or structural similarity to barnacle adhesive.

CONCLUSIONS

Alternating binary patterns of charge throughout homologous cementproteins from the barnacle adhesive were found to confer a unique foldedsecondary structure that enables polymerization of downstream dormantcement sequences. These dormant peptides only polymerize in the presenceof an anti-parallel structure, highlighting molecular recognition as akey mechanism in the formation of amyloid-like adhesives produced by thebarnacle. The invention demonstrates that the structures produced bypatterned cement sequences, and the progression of domain interactions,are critical in polymerizing materials that resemble the naturaladhesive. The sequences of the invention define a basic syntax used bythe barnacle to fabricate its adhesive, but also add new functions tothe growing language of materials formation through simple non-chargedamino acid sequences.

It will, of course, be appreciated that the above description has beengiven by way of example only and that modifications in detail may bemade within the scope of the present invention.

Throughout this application, various patents and publications have beencited. The disclosures of these patents and publications in theirentireties are hereby incorporated by reference into this application,in order to more fully describe the state of the art to which thisinvention pertains.

The invention is capable of modification, alteration, and equivalents inform and function, as will occur to those ordinarily skilled in thepertinent arts having the benefit of this disclosure. While the presentinvention has been described with respect to what are presentlyconsidered the preferred embodiments, the invention is not so limited.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the description provided above.

What is claimed:
 1. A peptide comprising an amino acid sequence as setforth in any one of SEQ ID Nos:1-8.
 2. The peptide of claim 1, whereinthe peptide has an amino acid sequence selected from the groupconsisting of SEQ ID No:2, SEQ ID No:5, and SEQ ID No:6.
 3. The peptideof claim 2, wherein the peptide aggregates to form fibrils.
 4. Thepeptide of claim 1, wherein the peptide has an amino acid sequenceselected from the group consisting of SEQ ID No:1, SEQ ID No:3, SEQ IDNo:4, SEQ ID No:7, and SEQ ID No:8.
 5. The peptide of claim 4, whereinpeptide does not aggregate.